Comparison of Physicochemical and Bioactive Properties of Turkish and New Zealand Manuka Honey

Ufuk Alpat Özge Erdem, İsmail Emir Akyıldız, İlknur Coşkun, Tuğçe Daştan, Sezer Acar, Özlem Cengiz, Ayça Karakuş, İlknur Temli Balparmak Research Center - İstanbul/Türkiye What is Pine Honey?

Pine Honey is a type of honeydew honey which is produced by honeybees that collect honeydew (sugary secretions) of an insect known as , which lives on certain pine trees.

Apimondia Congress / Montréal / Canada 9-12 September 2019 2 What is Pine Honey?

When the insects feed, they excrete waste in the form of honeydew. And honeybees collect this sweet liquid instead of flower and turn it into honey.

Apimondia Congress / Montréal / Canada 9-12 September 2019 3 The Actors of Pine Honey? Pine Honey is called twice refined honey The main actors of Pine Honey;

Pinus pinea and Pinus Brutia

Apis mellifera

Marchalina hellenica 4 What is Manuka Honey?

Mānuka honey is the monofloral product of Leptospermum scoparium (Myrtaceae) , a New Zealand native plant, said to possess ‘non-peroxide anti-bacterial activity’

Apimondia Congress / Montréal / Canada 9-12 September 2019 5 What is Manuka Honey?

It is described as having the Aroma; damp earth, heather aromatic and the Flavour; mineral, slightly bitter, tangy by the Bee Products Standards Council of New Zealand

Apimondia Congress / Montréal / Canada 9-12 September 2019 6 Sample Origins

Turkish Pine Honey is harvested from Southwestern pine forests of Turkey. Annual rainfall is above 2000 mm. Monthly average temperature is between 18-24°C, all during the winter, while summers are with exceeding temperatures of 35-40°C, allowing bees to rationally collect honeydew.

Apimondia Congress / Montréal / Canada 9-12 September 2019 7 Sample Origins

The native New Zealand Manuka tree grows predominantly on the North Island of New Zealand. To crop Manuka honey, beekeepers move their hives into areas where the Manuka is the predominant vegetation type when the Manuka is starting to flower, typically spring to early summer depending on the region within New Zealand. According to the Ministry for Primary Industries of New Zealand, Most of the Manuka Honey production is supplied by the North Island part.

Apimondia Congress / Montréal / Canada 9-12 September 2019 8 The Data Source of This Study

The results given here are from "The Accredited Analysis Results Database" of Balparmak Research and Development Center of Altıparmak Food Co. from Turkey

Apimondia Congress / Montréal / Canada 9-12 September 2019 9 Evaluated Properties

In this study, samples were evaluated for their; Physicochemical Properties; *Color *Moisture *Electrical Conductivity *Free Acidity *Diastase Activity *Invertase Activity *HMF *Proline * Profile *Total

Apimondia Congress / Montréal / Canada 9-12 September 2019 10 Evaluated Properties

Melissopalynological Properities; *Pollen Spectrum and; Bioactive Properties; *Mineral Content (Cr, Zn, Mn, Ca, Na, K, Co, Mo, Sn, Mg, Cu, Fe) *Antimicrobial Activity *Phenolic Compound Profile (43 compounds)

Apimondia Congress / Montréal / Canada 9-12 September 2019 11 Evaluated Properties

Analysis Method Equipment Moisture AOAC 969.38 Refractometer Electrical Conductivity DIN 10753 Conductivitymeter Hydroxymethylfurfural DIN 10751-3 HPLC-PDA Proline DIN-10754 Spectrophotometer Sugar profile DIN 10758 HPLC-RID Total disaccharides In-House procedure HPLC-RID Color AOAC 985.25 Tintometer Antimicrobial Activity* CLSI method (9th edition) Spectrophotometer Pollen Spectrum DIN 10760:2002-05 Microscope Invertase Activity IHC-2009 Spectrophotometer Free Acidity Titrimetric Titrimetric Diastase Activity In-House procedure Colorimetric Mineral Content In-House procedure ICP-MS Phenolic Profile In-House procedure LC-MSMS

*CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. CLSI document M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.

Apimondia Congress / Montréal / Canada 9-12 September 2019 12 RESULTS

Apimondia Congress / Montréal / Canada 9-12 September 2019 13 Physicochemical Properties

106,1 120,0

100,0

75,9 80,0

60,0

40,0 17,7 24,3 24,7 20,0 16,2 22,9 12,2

0,0 18,7 3,8 Colour (mm) Moisture (%) Free acidity (meq/kg) HMF (mg/kg) Diastase (DN)

Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 14 Physicochemical Properties

1200,0

1.028,2 1000,0

800,0 561,0

600,0 435,4 639,7

400,0 51,6

137,6 200,0

0,0

invertase (IU) Conductivity (μS/cm) Proline (mg/kg) Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 15 Physicochemical Properties-Sugar Profile

68,5 70,0 1,28 60,0 58,1 1,24 50,0 38,4

40,0 30,1 32,1

30,0 26,1

20,0

10,0 6,8 10,3 0,0

Fructose (%) Glucos (%) F+G (%) Total disaccharides (%) F/G

Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 16 Physicochemical Properties-Sugar Profile

2,0 2,0

1,8 1,4 1,5 1,7 1,6 1,7 1,5 1,4

1,2

1,0

0,8

0,6 0,5 0,4 0,4 0,0 0,4 0,2 0,0 0,0 0,0 0,0 (%) 0,1 0,0 (%) Turanose (%) (%) 0,0 Isomaltose (%) Erlose (%) Maltotriose (%) Melesitose (%) Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 17 Melissopalynological Profile

All the samples have been analyzed for their pollen spectrum. They all showed pollen spectrums consistent with their known origins.

Apimondia Congress / Montréal / Canada 9-12 September 2019 18 Functional Properties-Mineral Content

70 61,3

60 52,9 50 44,0 59,1

40

34,4 30 15,4 24,9 20 28,4 10,7 19,4 10 5,8 10,6 13,6 3,9 0

Chromium Calcium (μg/kg) Sodium (mg/kg) Cobalt (mg/kg) Molybdenum (μg/kg) Tin (μg/kg) (μg/kg) Magnessium (mg/kg)

Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 19 Functional Properties-Mineral Content

2,00% 1,99%

1,50%

1,08% 1,00%

0,50%

0,27% 0,19% 0,22% 0,13% 0,13% 0,08%0,09% 0,03% 0,08% 0,00% 0,02% Chromium Calcium (mg/kg) Sodium (mg/kg) Cobalt (μg/kg) Molybdenum Tin (μg/kg) Magnessium (μg/kg) (μg/kg) (mg/kg)

Pine Honey %RDA/Portion Manuka Honey %RDA/Portion

*RI= Reference Intake is the maximum recommended amount that an adult should eat in one day. This figure was previously known as the RDA (Recommended Daily Amount).

Apimondia Congress / Montréal / Canada 9-12 September 2019 20 Functional Properties-Mineral Content

4000,0 3739,7 3500,2 3500,0 2716,1 3000,0

2500,0 1596,5

2000,0 1444,4 1796,3 1500,0 769,4

1000,0 684,1

500,0

0,0 350,3 Zinc (μg/kg) 158,5 Manganese (μg/kg) Potasium (mg/kg) Copper (μg/kg) Iron (μg/kg) Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 21 Functional Properties-Mineral Content 3,12% 3,00%

2,63% 2,50%

2,00%

1,50%

1,16% 1,00%

0,77% 0,58% 0,50% 0,51%

0,26% 0,14%0,16% 0,13% 0,00% Zinc (μg/kg) Manganese (μg/kg) Potasium (mg/kg) Copper (μg/kg) Iron (μg/kg)

Pine Honey %RDA/Portion Manuka Honey %RDA/Portion

*RI= Reference Intake is the maximum recommended amount that an adult should eat in one day. This figure was previously known as the RDA (Recommended Daily Amount).

Apimondia Congress / Montréal / Canada 9-12 September 2019 22 Functional Properties- Antimicrobial Activity

15,6%

16,0%

14,0%

12,0%

10,0% 7,9% 6,0%

8,0%

6,0%

4,5% 4,0%

2,0%

0,0%

MIC against E.Coli MIC against Staph

Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 23 Functional Properties- Phenolic Profile • Undetected Phenolic Compounds

1. Cinnamyl Aldehyde 15.CAPE (caffeic acid phenethyl ester) 2. Phloroglucinol 16.Acacetin 3. 2-OH Cinnamic Acid 17.Kaempferol 4. Phenyllactic Acid 18.Luteolin 5. Gallic Acid 19.Epicatechin 6. Shikimic Acid 20.Catechin 7. Methylsyringate 21.Quercetin 8. Resveratrol 22.Ellagic Acid 9. Chrysin 23.Hesperetin 10.Pinocembrin 24.Isorhamnetin 11.Apigenin 25.Myricetin 12.Genistein 26.Rosmarinic Acid 13.Galangin 27.Rutin 14.Naringenin

Apimondia Congress / Montréal / Canada 9-12 September 2019 24 Functional Properties- Phenolic Profile 18,0

16,4 16,0

14,0

12,0

10,0

8,0

6,0 5,5 4,6 4,9

4,0 2,6 1,9 1,7 1,9 2,0 1,2 1,2 1,7 1,5 1,1 1,2 0,8 1,1 0,7 0,1 0,9 0,1 0,6 0,1 0,5 0,6 0,1 0,0 0,0 0,0 0,0 0,5 0,1 0,0 0,0 0,0

0,0

4-HydroxybenzoicAcid Vanilic Acid 3,4 Dimethoxy 3,4 Acid Transcinnamic Acid Protocatechunic Acid p-Coumaric m-Coumaric Acid Acid Homogentisic Caffeic Acid Acid Quinic Acid Ferrulic Acid Syringic Cinnamic Dimethoxy 3,4 Pinobanksin Epigallocatechin Taxifolin Acid Chlorgenic

Benzaldehyde Acid

Manuka Honey Pine Honey Apimondia Congress / Montréal / Canada 9-12 September 2019 25 Functional Properties- Phenolic Profile

97,3

100,0

90,0 63,4 80,0

70,0

60,0

50,0

40,0

30,0

20,0 4,4 10,0 8,3 0,0

Gallic Acid

Methylsyringate

Pine Honey Manuka Honey

Apimondia Congress / Montréal / Canada 9-12 September 2019 26 CONCLUSION

In this study 30 Pine Honey samples from Turkey and 15 Manuka Honey samples from New Zealand were analyzed.

Turkish Pine Honey’s and New Zealand Manuka Honey’s General and Bioactive Properties were determined by evaluating these analysis results. The most important outcomes of this study are given below:

➢ This study showed that New Zealand Manuka Honey is darker than Turkish Pine Honey. Moisture and Free acidity values did not show any significant differences. ➢ The HMF values of Manuka Honeys were 6 times over than Turkish Pine Honey’s HMF values, and average Diastase values of both varieties differ by 6 DNs. This can be due to the aging proces applied to some Manuka Honeys.

Apimondia Congress / Montréal / Canada 9-12 September 2019 27 CONCLUSION

The average electrical conductivity was 1.028,2 μS/cm for Turkish Pine Honey and for New Zealand Manuka Honey it was 561 μS/cm. Both honey types showed similar sugar profiles with some changes on «Sum of and (F+G), Isomaltose and Erlose). Where Turkish Pine Honey has lower F+G values but higher Isomaltose and Erlose values than Manuka honeys. Turkish Pine Honey showed two times higher cobalt content than New Zealand Manuka Honey which gives 2% of the RDI for one portion of honey (15 g). While New Zealand Manuka Honey had two times high sodium content than Turkish Pine Honey giving 0,03% of the RDI for one portion of honey (15 g).

Apimondia Congress / Montréal / Canada 9-12 September 2019 28 CONCLUSION

New Zealand Manuka Honey had five times high manganesse content than Turkish Pine Honey resulting 2,6% of the RDI for one portion of honey and Turkish Pine Honey showed 24 times higher iron content than New Zealand Manuka Honey which gives 3,1% of the RDI for one portion of honey. Turkish Pine Honey showed two times lower Minimum Inhibotary Content (MIC) than New Zealand Manuka Honey against E.Coli and about 1,3 times lower Minimum Inhibotary Content (MIC) against Staphylococcus Aereus.

Apimondia Congress / Montréal / Canada 9-12 September 2019 29 CONCLUSION

27 out of 43 phenolic compounds analyzed in this study were absent for both honey types. Turkish Pine Honey samples showed 4 times higher Protocatechunic Acid content, 2,5 times higher 4-Hydroxybenzoic Acid while New Zealand manuka Honey showed 3 times higher Homogentisic Acid content. Manuka honeys analyzed in this study showed very high amounts of Gallic Acid and Methylsyringate. For gallic acid this is not a very common outcome regarding the literature.

Based on these outcomes we may say that both honey types has their pros and cons and Turkish Pine Honey and New Zealand Manuka Honey are equally valuable honey types.

Apimondia Congress / Montréal / Canada 9-12 September 2019 30 Thank You For Your Kind Attention

Apimondia Congress / Montréal / Canada 9-12 September 2019 31